Uptake of external sulfate from the environment and use of internal vacuolar sulfate pools are two important aspects of the acquisition of sulfur for metabolism. In this study, we demonstrated that the vacuolar SULTR4-type sulfate transporter facilitates the efflux of sulfate from the vacuoles and plays critical roles in optimizing the internal distribution of sulfate in Arabidopsis thaliana. SULTR4;1-green fluorescent protein (GFP) and SULTR4;2-GFP fusion proteins were expressed under the control of their own promoters in transgenic Arabidopsis. The fusion proteins were accumulated specifically in the tonoplast membranes and were localized predominantly in the pericycle and xylem parenchyma cells of roots and hypocotyls. In roots, SULTR4;1 was constantly accumulated regardless of the changes of sulfur conditions, whereas SULTR4;2 became abundant by sulfur limitation. In shoots, both transporters were accumulated by sulfur limitation. Vacuoles isolated from callus of the sultr4;1 sultr4;2 double knockout showed excess accumulation of sulfate, which was substantially decreased by overexpression of SULTR4;1-GFP. In seedlings, the supplied [ 35 S]sulfate was retained in the root tissue of the sultr4;1 sultr4;2 double knockout mutant. Comparison of the double and single knockouts suggested that SULTR4;1 plays a major role and SULTR4;2 has a supplementary function. Overexpression of SULTR4;1-GFP significantly decreased accumulation of [ 35 S]sulfate in the root tissue, complementing the phenotype of the double mutant. These results suggested that SULTR4-type transporters, particularly SULTR4;1, actively mediate the efflux of sulfate from the vacuole lumen into the cytoplasm and influence the capacity for vacuolar storage of sulfate in the root tissue. The efflux function will promote rapid turnover of sulfate from the vacuoles particularly in the vasculature under conditions of lowsulfur supply, which will optimize the symplastic (cytoplasmic) flux of sulfate channeled toward the xylem vessels.

Cation diffusion facilitator (CDF) proteins belong to a family of heavy metal efflux transporters that might play an essential role in homeostasis and tolerance to metal ions. We investigated the subcellular localization of Arabidopsis thaliana AtMTP1, a member of the CDF family, and its physiological role in the tolerance to Zn using MTP1-deficient mutant plants. AtMTP1 was immunochemically detected as a 43 kDa protein in the vacuolar membrane fractioned by sucrose density gradient centrifugation. The expression level of AtMTP1 in suspension-cultured cells was not affected by the Zn concentration in the medium. When AtMTP1 fused with green fluorescent protein was transiently expressed in protoplasts prepared from Arabidopsis suspension-cultured cells, green fluorescence was clearly observed in the vacuolar membrane. A T-DNA insertion mutant line for AtMTP1 displays enhanced sensitivity to high Zn concentrations ranging from 200 to 500 microM, but not to Zn-deficient conditions. Mesophyll cells of the mtp1-1 mutant plants grown in the presence of 500 microM Zn were degraded, suggesting that Zn at high concentrations causes serious damage to leaves and that AtMTP1 plays a crucial role in preventing this damage in plants. Thus we propose that AtMTP1 is localized in the vacuolar membrane and is involved in sequestration of excess Zn in the cytoplasm into vacuoles to maintain Zn homeostasis.

A large number of proteins in the tonoplast, including pumps, carriers, ion channels and receptors support the various functions of the plant vacuole. To date, few proteins involved in these activities have been identified at the molecular level. In this study, proteomic analysis was used to identify new tonoplast proteins. A primary requirement of any organelle analysis by proteomics is that the purity of the isolated organelle needs to be high. Using suspension-cultured Arabidopsis cells (Arabidopsis Col-0 cell suspension), a method was developed for the isolation of intact highly purified vacuoles. No plasma membrane proteins were detected in Western blots of the isolated vacuole fraction, and only a few proteins from the Golgi and endoplasmic reticulum. The proteomic analysis of the purified tonoplast involved fractionation of the proteins by SDS-PAGE and analysis by LC-MS/MS. Using this approach, it was possible to identify 163 proteins. These included well-characterized tonoplast proteins such as V-type H+ -ATPases and V-type H+ -PPases, and others with functions reasonably expected to be related to the tonoplast. There were also a number of proteins for which a function has not yet been deduced.

The intracellular membrane dynamics of Arabidopsis cells under high salt treatment were investigated. When Arabidopsis was treated with high levels of NaCl in hydroponic culture, root tip cells showed rapid changes in the vacuolar volume, a decrease in the number of small acid compartments, active movement of vesicles and accumulation of Na(+) both in the central vacuole and in the vesicles around the main vacuole observed with the Na(+)-dependent fluorescence of Sodium Green. Detailed observation of Arabidopsis suspension-cultured cells under high salt treatment showed a similar pattern of response to that observed in root tip cells. Immunostaining of suspension-cultured cells with antibodies against AtNHX1 clearly showed the occurrence of dotted fluorescence in the cytoplasm only under salt treatment. We also confirmed the existence of AtNHX1 in the vacuolar membrane isolated from suspension-cultured cells with immunofluorescence. Knockout of the vacuolar Q(a)-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein VAM3/SYP22 caused an increase in salt tolerance. In mutant plants, the distribution of Na(+) between roots and shoots differed from that of wild-type plants, with Na(+) accumulating more in roots and less in the shoots of the mutant plants. The role of vesicle traffic under salt stress is discussed.